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dc.contributor.advisorJui-Hung Shienen_US
dc.contributor.authorSu, Yi-Daen_US
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dc.description.abstract家禽傳染性支氣管炎(Avian infectious bronchitis;IB)是由家禽傳染性支氣管炎病毒(Avian infectious bronchitis virus;IBV)感染雞隻造成呼吸道、泌尿道與生殖道疾病,常導致蛋雞產蛋率下降,肉雞換肉率下降與高死亡率,造成養雞業界嚴重經濟損失;牛傳染性下痢(Bovine infectious diarrhea;BID)是由牛冠狀病毒(Bovine coronavirus;BCoV)感染牛隻造成消化道疾病,常導致乳牛嚴重下痢與產乳量降低,亦造成養牛業界嚴重的經濟損失。冠狀病毒是單股正鏈的RNA病毒,其基因體長約30 kb,而且它的基因體結構跟真核細胞的mRNA相似,有5端非轉譯區、轉譯區、3端非轉譯區以及連接3端非轉譯區的poly(A) tail,先前研究已證實牛冠狀病毒感染HRT-18細胞株其poly(A) tail會隨時間改變而改變,為了探討冠狀病毒poly(A) tail長度的調節在雞與牛冠狀病毒中是否為共同的特徵,以及觀察在不同的宿主細胞是否也有poly(A) tail被調控的狀況,我們利用牛冠狀病毒(BCoV)感染另一種株化細胞BHK-21,以確定牛冠狀病毒(BCoV)的poly(A) tail是否也會隨著時間改變而改變。另外,我們也藉由雞冠狀病毒野外株(IBV-TW1)與疫苗株(IBV-H120)感染雞胚胎腎臟細胞和4日齡SPF小雞,以確定雞冠狀病毒(IBV)野外株或是疫苗株在初代細胞的感染過程中,其poly(A) tail是否也會被調控。結果顯示牛冠狀病毒的poly(A) tail長度在不同的細胞株也會被調節。此外,雞冠狀病毒的野外株以及疫苗株在初代細胞感染過程中,其poly(A) tail的長度在不同的時間也有不同的變化,顯示冠狀病毒的poly(A) tail長度的調控在感染的過程中是冠狀病毒共同的生物特徵,而且沒有宿主細胞的特異性。此外由結果可知,野外株(IBV-TW1)的poly(A) tail長度在感染細胞的過程當中比疫苗株(IBV-H120)的poly(A) tail長,因此推測冠狀病毒的poly(A) tail可能可以做為區別野外株(IBV-TW1)與疫苗株(IBV-H120)的標準之一。zh_TW
dc.description.abstractThe coronavirus genome is a single-stranded, (+)-sense RNA molecule of ~30 kilobases in length. As with a majority of eukaryotic messenger RNAs, the coronavirus genome contains a 5' cap, 5' and 3' untranslated regions of ~200 and ~300 nucleotides, respectively, and a poly(A) tail of >100 nt. We have found that the length of the poly(A) tail in the bovine coronavirus (BCoV), a group β coronavirus, is regulated throughout infection in human rectal tumor (HRT) cells and baby hamster kidney cell line BHK-21. To determine whether regulation of viral poly(A) tail length is a common feature of coronaviruses, and whether it is host-specific, poly(A) tail length of wild-type avian infectious bronchitis virus, strain TW1 (wt IBV-TW1) , a group γ coronavirus, was studied in the primary chicken embryo kidney(CEK) cells and 4-day-old SPF chickens. By ligating head-to-tail pyrophosphatase-treated viral RNA (+) strands and sequencing across the junction, the poly(A) tail length of wt IBV-TW1 was found to vary in CEK cells and in the trachea of the SPF chickens throughout infection, suggesting that regulation of poly(A) tail length is a common coronavirus phenomenon. Poly(A) tail length variation was similarly found with the IBV vaccine strain H-120 in CEK cells although the overall poly(A) tail length was shorter with this virus in comparison with the poly(A) tail length in IBV-TW1 virus. These results together suggest that the regulation of coronaviral poly(A) tail length during infection is a common feature among coronaviruses and is not host-specific. Furthermore, we speculate that the variation of poly(A) tail length may be an indicator of virulence based on the comparisons between wt IBV-TW1 (virulent) and IBV-H120 (avirulent).en_US
dc.description.tableofcontents中文摘要 i 英文摘要 ii 目次 iii 表次 vi 圖次 vii 第一章 緒言 1 第二章 文獻探討 2 第一節 雞傳染性支氣管炎之歷史背景 2 第二節 雞傳染性支氣管炎之特性 2 一、 臨床症狀 2 二、 自然宿主與傳播方式 3 第三節 雞傳染性支氣管炎病毒之特性 4 一、 病毒的種類 4 二、 病毒的型態與構造 4 三、 病毒的複製 5 四、 病毒的致病機制 5 第四節 雞傳染性支氣管炎之診斷方法 6 第五節 病毒poly(A) tail的形成以及在生活史上扮演的角色 7 一、 poly(A) tail在真核細胞messenger RNA的形成機轉與重要性 7 二、 病毒poly(A) tail的形成機轉與重要性 7 第三章 材料方法 9 第一節 病毒來源與增殖 9 一、病毒來源 9 二、細胞與實驗動物 9 三、病毒增殖 9 四、牛冠狀病毒感染BHK-21(baby hamster kidney cell)細胞株 10 第二節 病毒核酸之萃取與確認 10 第三節 引子的設計 10 第四節 反轉錄聚合酵素連鎖反應(RT-PCR) 11 一、反轉錄聚合酶鏈鎖反應產物確認 11 第五節 利用RNA ligase I及RT-PCR以確定IBV poly(A) tail的長度 11 一、消除genome頭端Cap的結構(Removal of the Cap from RNA) 11 二、酒精沉澱法 12 三、利用T4 RNA Ligase將病毒基因體5’端以及3’端相連接(T4 RNA Ligation) 12 五、反轉錄聚合酵素連鎖反應(cDNA合成與PCR) 12 六、反轉錄聚合酶鏈鎖反應產物確認 13 第六節 病毒分佈試驗 13 一、病毒接種與檢體的採集 13 二、檢體的處理和核酸的萃取 14 三、反轉錄聚合酵素連鎖反應(RT-PCR) 14 第四章 結果 15 第一節 病毒之分離與鑑定 15 第二節 病毒增殖 15 第三節 病毒力價測定 15 第四節 雞和牛冠狀病毒poly(A) tail在不同細胞上不同時間點的長度變化 15 一、牛冠狀病毒感染BHK-21其poly(A) tail的長度會被調節 15 二、雞冠狀病毒野外株(IBV-TW1)感染初代雞胚胎腎臟(CEK)細胞其poly(A) tail的長度會隨時間的改變而有所變化 16 三、雞冠狀病毒疫苗株(IBV-H120)感染初代雞胚胎腎臟(CEK)細胞其poly(A) tail會隨時間改變而改變 16 第五節 病毒分佈試驗 17 第六節 雞冠狀病毒poly(A) tail在活體上不同時間點的長度變化 17 一、雞冠狀病毒野外株(IBV-TW1)感染SPF雞其poly(A) tail會隨時間改變而改變 17 第五章 討論 27 第一節 牛冠狀病毒在感染HRT-18細胞和BHK-21細胞的過程當中其長度會有變 27 第二節 冠狀病毒poly(A) tail長度的變化也能發生在被病毒感染的初代細胞(primary cell culture)中 28 第三節 疫苗株(IBV-H120)poly(A) tail在初代雞胚胎腎臟(CEK)細胞中不同時間點的長度也有不同 28 第四節 雞冠狀病毒野外株(IBV-TW1)poly(A) tail在4日齡SPF雞隻其長度也會被調控 29 參考文獻 30zh_TW
dc.subjectcoronaviral poly(A) tailen_US
dc.title冠狀病毒Poly(A) Tail長度的調節並不具有宿主細胞的專一性zh_TW
dc.titleRegulation of Coronaviral Poly(A) Tail Length During Infection Is Not Host Cell-Specificen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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